The BEIP field-emission scanning transmission electron microscope has been used to obtain low-dose digital annular dark-field mass maps of isolated macromolecular assemblies, including the cytoskeleton and viruses. The technique has been applied to determine how the transport motor molecule, kinesin, mediates microtubule interactions in vitro. Maps of these structures provided information about the shapes and mass distributions of squid brain kinesin and taxol-stabilized microtubules. It was found that kinesins were loosely attached to the carbon support as asymmetrical dumbbell-shaped molecules, 40 to 52 nm long, with a mass of 379q15 kDa. The mass distribution and shape of kinesin suggest that these images represent the molecule in its shortened conformation. Kinesin microtubule complexes were organized as bundles of linearly arrayed microtubules, stitched together at irregular intervals by crossbridges (typically <25 nm long). The crossbridges had a mass similar to that of the isolated single molecule. The results suggest that kinesin has a second microtubule binding site, in addition to the known site on the motor domain of the heavy chain.